The ideal or preferred biomarker of breast density and/or cancer risk includes the following characteristics: (1) biologic plausibility; (2) a higher rate of expression in high-risk compared to low-risk populations; (3) an association with cancer in prospective studies; (4) expression minimally influenced by normal physiologic processes, or the ability to control for the influences of physiology; (5) the ability to obtain the biomarker using minimally invasive techniques at low costs; and (6) reproducibility. For purposes of the present invention the generally accepted National Institutes of Health definition of a “biomarker” is adopted: “a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” (Biomarkers Definitions working Group: Biomarkers And Surrogate Endpoints: Preferred Definitions And Conceptual Framework. Clin. Pharmacol. Ther. 2001; 69:89-95). As will be shown hereinbelow, the present invention relates generally to the measurement, especially the non-invasive measurement, of electrophysiological characteristics, preferably subepithelial impedance, as a biomarker for estimating breast density and further, the use of such estimated breast density as a further biomarker for breast cancer or abnormal tissue, preferably for use in assessing the risk that an individual will develop breast cancer or abnormal tissue.
The present invention relates generally to the detection of proliferative, abnormal or cancerous tissue, and more particularly, to the detection of changes in the electrophysiological characteristics of proliferative, abnormal or cancerous tissue and to changes in those electrophysiological characteristics related to the functional, structural and topographic (the interaction of shape, position and function) relationships of the tissue during the development of malignancy. These measurements can be made in the absence and presence of pharmacological and hormonal agents to reveal and accentuate the electrophysiological characteristics of proliferative, abnormal or cancerous tissue.
Cancer is a leading cause of death in both men and women in the United States. Difficulty in detecting proliferative, abnormal pre-cancerous or cancerous tissue before treatment options become non-viable is one of the reasons for the high mortality rate. Detecting of the presence of proliferative, abnormal or cancerous tissues is difficult, in part, because such tissues are largely located deep within the body, thus requiring expensive, complex, invasive, and/or uncomfortable procedures. For this reason, the use of detection procedures is often restricted until a patient is experiencing symptoms related to the abnormal tissue. Many forms of cancers or tumors, however, require extended periods of time to attain a detectable size (and thus to produce significant symptoms or signs in the patient). It is often too late for effective treatment by the time the detection is performed with currently available diagnostic modalities.
Breast cancer is the most common malignancy affecting women in the Western World. The reduction in mortality for this common disease depends on early detection. The mainstay of early detection are X-ray mammography and clinical breast examination. Both are fraught with problems of inaccuracy. For example, mammography has a lower sensitivity in women with dense breasts, and is unable to discriminate between morphologically similar benign or malignant breast lesions.
Clinical breast examinations are limited because lesions less than one cm are usually undetectable and larger lesions may be obscured by diffuse nodularity, fibrocystic change, or may be too deep in the breast to enable clinical detection. Patients with positive mammographic or equivocal clinical findings often require biopsy to make a definitive diagnosis. Moreover, biopsies may be negative for malignancy in up to 80% of patients.
Accordingly, mammography and clinical breast examination have relatively poor specificity in diagnosing breast cancer. Therefore many positive mammographic findings or lesions detected on clinical breast examination ultimately prove to be false positives resulting in physical and emotional trauma for patients. Improved methods and technologies to identify patients who need to undergo biopsy would reduce healthcare costs and avoid unnecessary diagnostic biopsies.
It is also desirable to develop improved technology suitable for characterizing pre-cancerous tissue and cancer in other tissue types and elsewhere in the body, particularly methods and devices suitable for ascertaining the condition of bodily ductal structures, e.g., the prostate, pancreas, etc., as well as the breast. Such characterization may ultimately be useful in diagnosis or risk assessment.
One proposed method for early detection of cancerous and pre-cancerous tissue includes measuring of the electrical impedance of biological tissue. For example, U.S. Pat. No. 3,949,736 discloses a low-level electric current passed through tissue, with a measurement of the voltage drop across the tissue providing an indirect indication of the overall tissue impedance. This method teaches that a change in impedance of the tissue is associated with an abnormal condition of the cells composing the tissue, indicating a tumor, carcinoma, or other abnormal biological condition. This disclosure, however, does not discuss either an increase or decrease in impedance associated with abnormal cells, nor does it specifically address tumor cells or other patient-specific factors that affect electrophysiological properties.
It is also noted that the above and similar systems do not consider DC electrical properties of the epithelium. Most common malignancies develop in an epithelium (the cell layer that lines a hollow organ, such as the bowel, or ductal structures such as the breast or prostate), that maintains a transepithelial electropotential. Early in the malignant process the epithelium loses its transepithelial potential, particularly when compared to epithelium some distance away from the developing malignancy. The combination of transepithelial electropotential measurements with impedance are more accurate in diagnosing pre-cancerous and cancerous conditions.
Another disadvantage of the above referenced system is that the frequency range of the electrical signal is not defined. Certain information is obtained about cells according to the range of frequencies selected. Different frequency bands may be associated with different structural or functional aspects of the tissue. See, for example, F. A. Duck, Physical Properties of Tissues, London: Academic Press, 2001; K. R. Foster, H. P. Schwan, Dielectric properties of tissues and biological materials: a critical review, Crit. Rev. Biomed. Eng., 1989, 17 (1): 25-104. For example at high frequencies such as greater than about 1 GHz molecular structure has a dominating effect on the relaxation characteristics of the impedance profile. Relaxation characteristics include the delay in the response of a tissue to a change in the applied electric field. For example, an applied AC current results in a voltage change across the tissue which will be delayed or phase shifted, because of the impedance characteristics of the tissue. Relaxation and dispersion characteristics of the tissue vary according to the frequency of the applied signal.
At lower frequencies, such as less than about 100 Hz, or the so called α-dispersion range, alterations in ion transport and charge accumulations at large cell membrane interfaces dominate the relaxation characteristics of the impedance profile. In the frequency range between a few kHz and about 1 MHz, or the so-called β-dispersion range, cell structure dominates the relaxation characteristics of the epithelial impedance profile. Within this range at low kHz frequencies, most of the applied current passes between the cells through the paracellular pathway and tight junctions. At higher frequencies in the β-dispersion range the current can penetrate the cell membrane and therefore passes both between and through the cells, and the current density will depend on the composition and volume of the cytoplasm and cell nucleus. Characteristic alterations occur in the ion transport of an epithelium during the process of malignant transformation affecting the impedance characteristics of the epithelium measured at frequencies in the α-dispersion range. Later in the malignant process, structural alterations with opening of the tight junctions and decreasing resistance of the paracellular pathways, together with changes in the composition and volume of the cell cytoplasm and nucleus, affect the impedance measured in the β-dispersion range.
Another disadvantage with the above referenced system is that the topography of altered impedance is not examined. By spacing the measuring electrodes differently the epithelium can be probed to different depths. The depth that is measured by two surface electrodes is approximately half the distance between the electrodes. Therefore electrodes 1 mm apart will measure the impedance of the underlying epithelium to a depth of approximately 500 microns. It is known, for example, that the thickness of bowel epithelium increases at the edge of a developing tumor to 1356±208μ compared with 716±112μ in normal bowel. D. Kristt, et al., Patterns of proliferative changes in crypts bordering colonic tumors: zonal histology and cell cycle marker expression, Pathol. Oncol. Res 1999; 5 (4): 297-303. Thickening of the ductal epithelium of the breast is also observed as ductal carcinoma in-situ develops. By comparing the measured impedance between electrodes spaced approximately 2.8 mm apart and compared with the impedance of electrodes spaced approximately 1.4 mm apart, information about the deeper and thickened epithelium may be obtained. See, for example, L. Emtestam, S. Ollmar, Electrical impedance index in human skin: measurements after occlusion, in 5 anatomical regions and in mild irritant contact dermatitis, Contact Dermatitis 1993; 28 (2): 104-108.
A further disadvantage of the above referenced methods is that they do not probe the specific conductive pathways that are altered during the malignant process. For example, potassium conductance is reduced in the surface epithelium of the colon early in the malignant process. By using electrodes spaced less than 1 mm apart with varying concentrations of potassium chloride the potassium conductance and permeability may be estimated in the surface epithelium at a depth from less than 500μ to the surface.
A number of non-invasive impedance imaging techniques have been developed in an attempt to diagnose breast cancer. Electrical impedance tomography (EIT) is an impedance imaging technique that employs a large number of electrodes placed on the body surface. The impedance measurements obtained at each electrode are then processed by a computer to generate a 2-dimensional or 3-dimensional reconstructed tomographic image of the impedance and its distribution in 2 or 3 dimensions. This approach relies on the differences in conductivity and impedivity between different tissue types and relies on data acquisition and image reconstruction algorithms which are difficult to apply clinically.
The majority of EIT systems employ “current-driving mode,” which applies a constant AC current between two or more current-passing electrodes, and measures the voltage drop between other voltage-sensing electrodes on the body surface. Another approach is to use a “voltage-driving approach,” which applies a constant AC voltage between two or more current-passing electrodes, and then measures the current at other current-sensing electrodes. Different systems vary in the electrode configuration, current or voltage excitation mode, the excitation signal pattern, and AC frequency range employed.
Another disadvantage with using EIT to diagnose breast cancer is the inhomogeneity of breast tissue. The image reconstruction assumes that current passes homogeneously through the breast tissue which is unlikely given the varying electrical properties of different types of tissue comprising the breast. In addition image reconstruction depends upon the calculation of the voltage distribution on the surface of the breast from a known impedance distribution (the so called forward problem), and then estimating the impedance distribution within the breast from the measured voltage distribution measured with surface electrodes (the inverse problem). Reconstruction algorithms are frequently based on finite element modeling using Poisson's equation and with assumptions with regard to quasi-static conditions, because of the low frequencies used in most EIT systems.
Other electrically-based methods for cancer diagnosis are disclosed in the patent and journal literature. A brief discussion of such disclosures can be found in the copending patent application by the inventor herein, U.S. Ser. No. 11/879,805, filed Jul. 18, 2007, the disclosure of which is incorporated herein by reference.
Another potential source of information for the detection of abnormal tissue is the measurement of transport alterations in the epithelium. Epithelial cells line the surfaces of the body and act as a barrier to isolate the body from the outside world. Not only do epithelial cells serve to insulate the body, but they also modify the body's environment by transporting salts, nutrients, and water across the cell barrier while maintaining their own cytoplasmic environment within fairly narrow limits. One mechanism by which the epithelial layer withstands the constant battering is by continuous proliferation and replacement of the barrier. This continued cell proliferation may partly explain why more than 80% of cancers are of epithelial cell origin. Moreover, given their special abilities to vectorially transport solutes from blood to outside and vice versa, it appears that a disease process involving altered growth regulation may have associated changes in transport properties of epithelia.
Epithelial cells are bound together by tight junctions, which consist of cell-to-cell adhesion molecules. These adhesion proteins regulate the paracellular transport of molecules and ions between cells and are dynamic structures that can tighten the epithelium, preventing the movement of substances, or loosen allowing substances to pass between cells. Tight junctions consist of integral membrane proteins, claudins, occludins and JAMs (junctional adhesion molecules). Tight junctions will open and close in response to intra and extracellular stimuli.
A number of substances will open or close tight junctions. The pro-inflammatory agent TGF-alpha, cytokines, IGF and VEGF opens tight junctions. Zonula occludens toxin, nitric oxide donors, and phorbol esters also reversibly open tight junctions. Other substances close tight junctions including calcium, H2 antagonists and retinoids. Various hormones such as prolactin and glucocorticoids will also regulate the tight junctions. Other substances added to drug formulations act as non-specific tight junction modulators including chitosan and wheat germ agglutinin.
The above referenced substances and others may act directly or indirectly on the tight junction proteins, which are altered during carcinogenesis. For example claudin-7 is lost in breast ductal epithelium during the development of breast cancer. The response of the tight junctions varies according to the malignant state of the epithelium and their constituent proteins. As a result the opening or closing of tight junctions is affected by the malignant state of the epithelium.
Surface measurements of potential or impedance are not the same as measurements performed across the breast epithelium where electrical contact is made between the luminal surface of the duct and the overlying skin. Transepithelial depolarization is an early event during carcinogenesis, which may affect a significant region of the epithelium (a “field defect”). This depolarization is accompanied by functional changes in the epithelium including ion transport and impedance alterations. Early on in the process these take the form of increased impedance because of decreased specific electrogenic ion transport processes. As the tumor begins to develop in the pre-malignant epithelium, structural changes occur in the transformed cells such as a breakdown in tight junctions and nuclear atypia. The structural changes result in a marked reduction in the impedance of the tumor. As previously described by the present inventor, understanding and interpreting the pattern and gradient of electrical changes in the epithelium can assist in the diagnosis of cancer from a combination of DC electrical and impedance measurements.
Breast cancer is thought to originate from epithelial cells in the terminal ductal lobular units (TDLUs) of mammary tissue. These cells proliferate and have a functional role in the absorption and secretion of various substances when quiescent and may produce milk when lactating. Functional alterations in breast epithelium have largely been ignored as a possible approach to breast cancer diagnosis. Breast epithelium is responsible for milk formation during lactation. Every month pre-menopausal breast epithelium undergoes a “rehearsal” for pregnancy with involution following menstruation. The flattened epithelium becomes more columnar as the epithelium enters the luteal phase from the follicular phase. In addition, duct branching and the number of acini reach a maximum during the latter half of the luteal phase. Just before menstruation apoptosis of the epithelium occurs and the process starts over again unless the woman becomes pregnant.
It is known that various hormones affect breast epithelial ion transport. For example, prolactin decreases the permeability of the tight-junctions between breast epithelial cells, stimulates mucosal to serosal Na+ flux, upregulates Na+:K+:2Cl− cotransport and increases the [K+] and decreases the [Na+] in milk. Glucocorticoids control the formation of tight-junctions increasing transepithelial resistance and decreasing epithelial permeability. Administration of cortisol into breast ducts late in pregnancy has been shown to increase the [K+] and decrease [Na+] of ductal secretions. Progesterone inhibits tight-junction closure during pregnancy and may be responsible for the fluctuations in ductal fluid electrolytes observed during menstrual cycle in non-pregnant women, and discussed above. Estrogen has been observed to increase cell membrane and transepithelial potential and may stimulate the opening of K+-channels in breast epithelial cells. The hormones mentioned above vary diurnally and during menstrual cycle. It is likely that these variations influence the functional properties of breast epithelium altering the ionic concentrations within the lumen, the transepithelial potential and impedance properties, which are dependent upon the ion transport properties of epithelial cells and the transcellular and paracellular conductance pathways.
Breast cancer biomarkers have recently attracted national attention and various markers that have been studied in women at risk for breast cancer include the following:
Germline Mutations and Polymorphisms: Highly penetrant genes such as BRCA1/BRCA2 with deleterious germline mutations are strong predictors of breast cancer development, but are found in less than 5-10% of women with breast cancer and in only 1% of the general population. Single nucleotide polymorphisms of genes whose protein products are involved in carcinogen and hormone metabolism and/or DNA repair are associated with relative risks of 1.4-2.0, however combined polymorphisms may be associated with significantly higher relative risks.
Hormones and Metabolites: Serum bioavailable estradiol and testosterone may represent risk biomarkers in postmenopausal women, and serum insulin-like growth factor-I (IGF-I) and its binding protein-3 (IGFBP-3) in premenopausal women. However none have been established to definitively identify high-risk women.
Mammographic Breast density and Intraepithelial Neoplasia: Mammographic breast density and breast intra-epithelial neoplasia apply to many more of the female population than germline mutations in tumor-suppressor genes. Furthermore, since they are subject to modulation, these risk biomarkers might be used to monitor change in breast cancer susceptibility from a prevention intervention standpoint. Mammographic breast density and intra-epithelial neoplasia are useful in both pre- and post-menopausal women. However, Tice et al. (Breast Cancer Res. Treat. 94:115-22, 2005) and Chen et al. (J. Natl. Cancer Inst. 98:1215-26, 2006) have reported that mammographic density adds modestly to the Gail model (M. H. Gail et al., J. Natl. Cancer Inst. 81 (24): 1879-86, 1989) in improving discriminatory accuracy. Assessing density typically requires the use of radiation-based methods and is subject to inter-observer variability. Improvements in the estimation of breast density have been proposed using volumetric and three dimensional magnetic resonance imaging (MRI) approaches.
Breast intra-epithelial neoplasia is a risk biomarker with close biologic association with cancer, and is least likely to be affected by normal physiologic processes, although ductal proliferation may be influenced by position in menstrual cycle. (See Fabian et al., Endocr. Relat Cancer 12:185-213, 2005, for a review). This includes proliferative breast disease without atypia, atypical ductal and lobular hyperplasia and in situ cancer. Within the spectrum of intra-epithelial neoplasia, an increase in morphologic abnormality is associated with a progressive increase in relative risk and a shorter time (decreased latency) to the development of breast cancer. Proliferative breast disease without atypia (moderate to florid hyperplasia, sclerosing adenosis, papillomas, etc.) is found in approximately 25-30% of diagnostic biopsies and is associated with a 1.4-2.0-fold increase in the relative risk for breast cancer. Higher relative risks associated with proliferative disease without atypia (e.g. 2.0 versus 1.4) may be associated with older age (>50 years), because of a failure to down-regulate proliferation at menopause, or a positive family history.
Ductal or lobular atypical hyperplasia, identified on diagnostic biopsies, is associated with an approximate 5-fold increase in relative risk regardless of other risk factors. Women identified with atypia, but without a positive family history, have an approximately 4 to 5-fold increased risk, whereas women with a positive family history double their relative risk of breast cancer to approximately 10-fold. Atypical ductal and lobular hyperplasia are observed in 3-10% of unselected diagnostic surgical and core needle biopsies. Those women who ultimately develop cancer have a higher proportion of prior benign biopsies exhibiting atypical hyperplasia than those who do not. Several investigators have suggested that atypical hyperplasia may arise more commonly from an intermediate lesion called an unfolded lobule (A for ductal, B for lobular) than hyperplasia of the usual type (HUT). Both atypical hyperplasia and HUT may arise from unfolded lobules. These unfolded lobules are characterized by increased cellularity and proliferation with distension of the terminal lobule duct unit.
Genetic changes associated with Intraepithelial Neoplasia: ADH and DCIS often have similar molecular and genetic changes as assessed by immunocytology or mRNA gene profiles. Approximately 50% of ADH lesions demonstrate loss of heterozygosity, which is observed somewhat less frequently for HUT lesions. The most frequent chromosomal losses are at 16q and 17p for both HUT and atypical hyperplasia, similar to those observed for DCIS. Similar chromosomal gains and losses for non-invasive and invasive lobular cancer are observed using comparative genomic hybridization techniques. The loss of 16q, which contains E-cadherin, a tumor-suppressor gene involved in cell adhesion and cell-cycle regulation. It is reported that E-cadherin is expressed in normal cells, but is lost in LCIS and invasive lobular cancer. ADH is reported in 5% or less of diagnostic biopsies, and has been reported in 9% of autopsy specimens from average-risk women. However, it is observed in 39% of prophylactic mastectomy specimens from high-risk women. Furthermore, it has been reported that 57% of women with a family history consistent with that of a mutation in BRCA1 and/or BRCA2 had atypical ductal or lobular lesions and/or in situ cancer and these lesions were often multifocal or multicentric.
Altered Hormonal receptor status: An inverse relationship has been observed between serum estradiol and ER-α of breast epithelium in women without breast cancer, which is dependent on position in menstrual cycle. This relationship has not been observed in breast epithelium derived from women with breast cancer. Epithelial proliferation was inversely correlated to ER in controls, but was positively related in breast cancer cases. These observations have lead to the suggestion that that the surrounding epithelium of women with breast cancer may display an aberrant response to estradiol with ER up-regulating in the luteal phase of menstrual cycle, whereas it down-regulates in breast epithelium from women without breast cancer. The effect of this aberrant response on breast epithelial morphology is unknown. Women with an increased risk appear not to down-regulate in their menstrual cycle as do normal risk women. Proliferative up-regulation may persist in women at increased risk for breast cancer based on the inventor's observations in women undergoing breast biopsy for proliferative breast disease.
Thus, there remains a need for effective and practical methods for characterizing tissue, particularly breast tissue and the density of the breast, that is susceptible to abnormal changes and for using such information to assess the risk that a patient, and particularly one that is substantially asymptomatic, will be found to have proliferative, abnormal or pre-cancerous breast tissue.
The disclosures of the following patent applications, each to Richard J. Davies, the inventor herein, are hereby incorporated by reference herein: U.S. patent application Ser. No. 11/879,805, filed Jul. 18, 2007, entitled “Method and System for Detecting Electrophysiological Changes in Pre-Cancerous and Cancerous Tissue” published as US 2008/0009764 Jan. 10, 2008; U.S. patent application Ser. No. 10/151,233, filed May 20, 2002, entitled “Method and System for Detecting Electrophysiological Changes in Pre-Cancerous and Cancerous Tissue,” now U.S. Pat. No. 6,922,586, issued Jul. 26, 2005; U.S. patent application Ser. No. 10/717,074, filed Nov. 19, 2003, entitled “Method And System For Detecting Electrophysiological Changes In Pre-Cancerous And Cancerous Breast Tissue And Epithelium”; and U.S. patent application Ser. No. 10/716,789, filed Nov. 19, 2003, published as US 2004/0253652 Dec. 16, 2004, entitled “Electrophysiological Approaches To Assess Resection and Tumor Ablation Margins and Responses To Drug Therapy” published as US 2004/0152997 Aug. 5, 2004.